Genetically modified non-human animals with humanized immunoglobulin and mhc loci

Abstract

Genetically modified animals and cells with humanized light chain immunoglobulin locus and/or humanized heavy chain immunoglobulin locus. The animals and cells can also express a human or chimeric (e.g., humanized) major histocompatibility complex (MHC) protein complex.

What is claimed is: 
     
         1 . A genetically-modified non-human animal comprising at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and can undergo VDJ rearrangement, wherein the animal expresses a fusion protein comprising β2 microglobulin (B2M) and a human or humanized major histocompatibility complex (MHC) α chain. 
     
     
         2 . The animal of  claim 1 , wherein the animal comprises at least 150 human IGHV genes selected from Table 1, at least 20 human IGHD genes selected from Table 2, and at least 5 human IGHJ genes selected from Table 3. 
     
     
         3 . The animal of  claim 1 , wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human subject. 
     
     
         4 . The animal of  claim 1 , wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human cell. 
     
     
         5 . The animal of  claim 1 , wherein the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin gene locus. 
     
     
         6 . The animal of  claim 5 , wherein the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and/or one or more mouse IGHJ genes in Table 6. 
     
     
         7 . The animal of  claim 5 , wherein the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of a contiguous sequence starting from mouse IGHV1-85 gene to mouse IGHJ4 gene. 
     
     
         8 . The animal of  claim 1 , wherein the animal comprises one or more endogenous IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes. 
     
     
         9 . The animal of  claim 1 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus, wherein the unmodified human sequence is at least 800 kb. 
     
     
         10 . The animal of  claim 1 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV1-2. 
     
     
         11 . The animal of  claim 1 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV6-1. 
     
     
         12 . The animal of  claim 1 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHD1-1 to human IGHJ6. 
     
     
         13 . The animal of  claim 1 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHJ6. 
     
     
         14 . The animal of  claim 1 , wherein the animal is homozygous with respect to the heavy chain immunoglobulin gene locus. 
     
     
         15 . The animal of  claim 1 , wherein the animal is heterozygous with respect to the heavy chain immunoglobulin gene locus. 
     
     
         16 . The animal of  claim 1 , wherein the animal further comprises at an endogenous light chain immunoglobulin gene locus, one or more human IGKV genes, and one or more human IGKJ genes. 
     
     
         17 . The animal of  claim 1 , wherein the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus. 
     
     
         18 . The animal of  claim 1 , wherein the animal is a rodent (e.g., a mouse). 
     
     
         19 . A genetically-modified non-human animal comprising at an endogenous light chain immunoglobulin gene locus, one or more human IGKV genes and one or more human IGKJ genes, wherein the animal expresses a fusion protein comprising β2 microglobulin (B2M) and a human or humanized major histocompatibility complex (MHC) α chain. 
     
     
         20 . The animal of  claim 19 , wherein the animal comprises all human IGKV genes in Table 7, and all human IGKJ genes in Table 8. 
     
     
         21 . The animal of  claim 19 , wherein the animal comprises an unmodified sequence derived from a human light chain immunoglobulin gene locus starting from human IGKV3D-7 to human IGKJ5. 
     
     
         22 . The animal of  claim 19 , wherein the animal comprises a disruption in the animal's endogenous light chain immunoglobulin gene locus. 
     
     
         23 . The animal of  claim 19 , wherein the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of one or more mouse IGKV genes in Table 9 and one or more mouse IGKJ genes in Table 10. 
     
     
         24 . The animal of  claim 22 , wherein the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of a sequence starting from mouse IGKV2-137 to mouse IGKJ5. 
     
     
         25 . The animal of  claim 19 , wherein the animal comprises an endogenous IGKC. 
     
     
         26 . The animal of  claim 19 , wherein the animal is homozygous with respect to the light chain immunoglobulin gene locus. 
     
     
         27 . The animal of  claim 19 , wherein the animal is heterozygous with respect to the light chain immunoglobulin gene locus. 
     
     
         28 . The animal of  claim 19 , wherein the animal further comprises at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. 
     
     
         29 . The animal of  claim 19 , wherein the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus. 
     
     
         30 . The animal of  claim 19 , wherein the animal is a rodent (e.g., a mouse). 
     
     
         31 . A genetically-modified non-human animal comprising at the endogenous light chain immunoglobulin locus, an exogenous light chain variable region gene sequence, wherein the exogenous light chain variable region gene sequence comprises no more than three human IGKV genes and no more than two human IGKJ genes, wherein the no more than three human IGKV genes and the no more than two human IGKJ genes are operably linked to an endogenous light chain constant domain gene, wherein the animal expresses a fusion protein comprising β2 microglobulin (B2M) and a human or humanized major histocompatibility complex (MHC) α chain. 
     
     
         32 . The animal of  claim 31 , wherein the no more than three human IGKV genes are selected from Table 7, and the no more than two human IGKJ genes are selected from Table 8. 
     
     
         33 . The animal of  claim 31 or 32 , wherein the exogenous light chain variable region gene sequence comprises one human IGKV gene and one human IGKJ gene. 
     
     
         34 . The animal of any one of  claims 31-33 , wherein the exogenous light chain variable region gene sequence further comprises a human IGKJ 3′-UTR sequence. 
     
     
         35 . The animal of any one of  claims 31-34 , wherein the exogenous light chain variable region gene in one or more cells of the animal can subject to somatic hypermutations. 
     
     
         36 . The animal of  claim 35 , wherein the somatic hypermutations can result in up to one, two, or three amino acid changes in light chain variable regions in the one or more cells of the animal. 
     
     
         37 . The animal of any one of  claims 31-36 , wherein exogenous light chain variable region gene sequence comprises one human IGKV gene and one human IGKJ gene, wherein the human IGKV gene is selected from the group consisting of IGKV3-20, IGKV3-11, and IGKV1-39, wherein the human IGKV gene and the human IGKJ gene are operably linked. 
     
     
         38 . The animal of  claim 37 , wherein the human IGKV gene is IGVK3-11. 
     
     
         39 . The animal of any one of  claims 31-38 , wherein the human IGKJ gene is selected from the group consisting of IGKJ1 and IGKJ4. 
     
     
         40 . The animal of any one of  claims 31-39 , wherein the human IGKV gene is IGKV1-39 and the human IGKJ gene is IGKJ4. 
     
     
         41 . The animal of any one of  claims 31-39 , wherein the human IGKV gene is IGKV3-11 and the human IGKJ gene is IGKJ1. 
     
     
         42 . The animal of any one of  claims 31-39 , wherein the human IGKV gene is IGKV3-20 and the human IGKJ gene is IGKJ1. 
     
     
         43 . The animal any one of  claims 31-42 , wherein the animal further comprises a promoter sequence that is operably linked to the human IGKV gene, wherein the promoter sequence is within 2500 or 3000 bp of the human IGKV gene. 
     
     
         44 . The animal of  claim 43 , wherein the promoter is an IGKV3-20 promoter, an IGKV3-11 promoter, or an IGKV1-39 promoter. 
     
     
         45 . The animal of any one of  claims 31-44 , wherein the animal comprises a disruption in the animal's endogenous light chain immunoglobulin gene locus. 
     
     
         46 . The animal of  claim 45 , wherein the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of one or more mouse IGKV genes in Table 9 and one or more mouse IGKJ genes in Table 10. 
     
     
         47 . The animal of  claim 46 , wherein the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of a sequence starting from mouse IGKV2-137 to mouse IGKJ5. 
     
     
         48 . The animal of any one of  claims 31-47 , wherein the animal comprises an endogenous IGKC. 
     
     
         49 . The animal of any one of  claims 31-48 , wherein the animal further comprises a kappa intronic enhancer 5′ with respect to the endogenous IGKC and/or a kappa 3′ enhancer. 
     
     
         50 . The animal of any one of  claims 31-49 , wherein the human light chain variable region is a rearranged sequence. 
     
     
         51 . The animal of any one of  claims 31-50 , wherein the animal is homozygous with respect to the light chain immunoglobulin gene locus. 
     
     
         52 . The animal of any one of  claims 31-50 , wherein the animal is heterozygous with respect to the light chain immunoglobulin gene locus. 
     
     
         53 . The animal of any one of  claims 31-52 , wherein the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus. 
     
     
         54 . The animal of any one of  claims 31-53 , wherein the animal is a rodent (e.g., a mouse). 
     
     
         55 . The animal of any one of  claims 31-54 , wherein the animal further comprises at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHKJ genes, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and can undergo VDJ rearrangement. 
     
     
         56 . The animal of  claim 55 , wherein the animal comprises at least 150 human IGHV genes selected from Table 1, at least 20 human IGHD genes selected from Table 2, and at least 5 human IGHJ genes selected from Table 3. 
     
     
         57 . The animal of  claim 55 , wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human subject. 
     
     
         58 . The animal of  claim 55 , wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human cell. 
     
     
         59 . The animal of any one of  claims 55-58 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus, wherein the unmodified human sequence is at least 800 kb. 
     
     
         60 . The animal of any one of  claims 55-59 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV1-2. 
     
     
         61 . The animal of any one of  claims 55-59 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV6-1. 
     
     
         62 . The animal of any one of  claims 55-61 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHD1-1 to human IGHJ6. 
     
     
         63 . The animal of any one of  claims 55-59 , wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHJ6. 
     
     
         64 . The animal of any one of  claims 1-63 , wherein the animal lacks an endogenous immunoglobulin heavy chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous heavy chain variable domain (e.g., a mouse heavy chain variable domain). 
     
     
         65 . The animal of any one of  claims 1-63 , wherein the animal lacks an endogenous immunoglobulin light chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous light chain variable domain (e.g., a mouse light chain variable domain). 
     
     
         66 . The animal of any one of  claims 1-65 , wherein the animal can produce a humanized antibody. 
     
     
         67 . The animal of any one of  claims 1-66 , wherein the genome of the animal comprises at least one chromosome comprising a sequence encoding the fusion protein. 
     
     
         68 . The animal of any one of  claims 1-67 , wherein the fusion protein comprises a human or humanized B2M protein. 
     
     
         69 . The animal of any one of  claims 1-68 , wherein the MHC α chain is a MHC class I α chain. 
     
     
         70 . The animal of any one of  claims 1-69 , wherein the MHC α chain is a chimeric MHC α chain. 
     
     
         71 . The animal of any one of  claims 1-69 , wherein the MHC α chain is a human HLA protein (e.g., HLA-A, HLA-B, or HLA-C). 
     
     
         72 . The animal of any one of  claims 1-69 , wherein the MHC α chain is a human HLA/mouse H-2 chimeric molecule, wherein the human HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, and wherein the mouse H-2 is selected from the group consisting of H-2K, H-2D, and H-2L. 
     
     
         73 . The animal of any one of  claims 1-69 , wherein the MHC α chain is a human HLA/mouse H2-D1 chimeric molecule. 
     
     
         74 . The animal of any one of  claims 1-69 , wherein the fusion protein comprises a human B2M protein and a chimeric MHC α chain comprising human HLA α1 and α2 domains. 
     
     
         75 . The animal of  claim 74 , wherein the chimeric MHC α chain further comprises a mouse H2-D1 α3 domain. 
     
     
         76 . The animal of any one of  claims 67-75 , wherein the sequence encoding the fusion protein is operably linked to an endogenous regulatory element (e.g., a promoter) at the endogenous β2 microglobulin (B2M) gene locus in the at least one chromosome. 
     
     
         77 . The animal of any one of  claims 71-76 , wherein the human HLA is human HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*0302, HLA-A*1101, HLA-A*2402, HLA-A*2901, HLA-A*3101, HLA-A*3201, HLA-A*3301, HLA-A*3303, HLA-B*4402, HLA-B*0702, HLA-C*0702, HLA-C*0102, HLA-C*0701, HLA-C*0401, HLA-C*0801, or HLA-C*0802. 
     
     
         78 . The animal of any one of  claims 1-70 , wherein the fusion protein comprises
 (a) a human B2M; and   (b) a human HLA (e.g., HLA-A, HLA-B, or HLA-C).   
     
     
         79 . The animal of  claim 78 , wherein the human B2M and the human HLA are linked via a linker peptide sequence. 
     
     
         80 . The animal of any one of  claims 1-70 , wherein the fusion protein comprises
 (a) a human B2M; and   (b) a chimeric MHC α chain.   
     
     
         81 . The animal of  claim 80 , wherein the human B2M and the chimeric MHC α chain are linked via a linker peptide sequence. 
     
     
         82 . The animal of  claim 80 or 81 , wherein the chimeric MHC α chain comprises human HLA α1 and α2 domains. 
     
     
         83 . The animal of  claim 82 , wherein the chimeric MHC α chain further comprises a human HLA α3 domain. 
     
     
         84 . The animal of  claim 82 , wherein the chimeric MHC α chain further comprises a MHC 3 domain endogenous to the animal and/or a MHC cytoplasmic region endogenous to the animal. 
     
     
         85 . The animal of any one of  claims 80-84 , wherein the chimeric MHC α chain comprises a α3 domain, a connecting peptide, a transmembrane region, and a cytoplasmic region of an endogenous MHC. 
     
     
         86 . The animal of any one of  claims 80-84 , wherein the animal is a mouse, and the chimeric MHC α chain comprises a α3 domain, a connecting peptide, a transmembrane region, and a cytoplasmic region of mouse H2-D1. 
     
     
         87 . The animal of any one of  claims 71-86 , wherein the fusion protein further comprises a signal peptide of the human HLA (e.g., at the N-terminus of the fusion protein). 
     
     
         88 . The animal of any one of  claims 1-87 , wherein the animal is heterozygous with respect to the sequence encoding the fusion protein. 
     
     
         89 . The animal of any one of  claims 1-87 , wherein the animal is homozygous with respect to the sequence encoding the fusion protein. 
     
     
         90 . A cell obtained from the animal of any one of  claims 1-89 . 
     
     
         91 . The cell of  claim 90 , wherein the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain that is derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region. 
     
     
         92 . The cell of  claim 90 , wherein the cell is a B cell that expresses a chimeric immunoglobulin light chain comprising an immunoglobulin light chain variable domain that is derived from a rearrangement of one or more human IGKV genes and one or more human IGKJ genes, and wherein the immunoglobulin light chain variable domain is operably linked to a non-human light chain constant region. 
     
     
         93 . The cell of  claim 90 , wherein the cell is an embryonic stem (ES) cell. 
     
     
         94 . A method of making an antibody that specifically binds to an antigen peptide-MHC complex, the method comprising
 exposing the animal of any one of  claims 1-89  to the antigen peptide-MHC complex comprising the antigen peptide.   
     
     
         95 . The method of  claim 94 , wherein the method further comprises
 producing a hybridoma from a cell collected from the animal; and   collecting or analyzing the antibody produced by the hybridoma.   
     
     
         96 . The method of  claim 94 or 95 , wherein the method further comprises sequencing the genome of the hybridoma. 
     
     
         97 . The method of any one of  claims 94-96 , wherein the antigen peptide is an antigen peptide (e.g., any one of the antigen peptides listed in Table 16). 
     
     
         98 . A method of obtaining a nucleic acid that encodes an antibody binding domain that specifically binds to an antigen peptide-MHC complex, the method comprising exposing the animal of any one of  claims 1-89  to the antigen peptide-MHC complex comprising the antigen peptide; and
 sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen peptide-MHC complex. 
 
     
     
         99 . A method of obtaining a sample, the method comprising
 exposing the animal of any one of  claims 1-89  to an antigen peptide-MHC complex; and   collecting the sample from the animal.   
     
     
         100 . The method of  claim 99 , wherein the sample is a spleen tissue, a lymphoid tissue, spleen cell, or a B cell. 
     
     
         101 . A method of screening an antibody that specifically binds to an antigen peptide-MHC complex, the method comprising
 exposing the animal of any one of  claims 1-89  to the antigen peptide-MHC complex comprising an antigen peptide of interest;   producing a hybridoma from a cell collected from the animal;   collecting or analyzing antibodies produced by the hybridoma;   incubating the antibodies with cells presenting the antigen peptide of interest, cells presenting a control antigen peptide and/or cells do not present any antigen peptide; and   determining that the antibodies can specifically bind to cells presenting the antigen peptide of interest, and optionally determining that the antibodies cannot bind to the cells presenting the control antigen peptide or the cells that do not present any antigen peptide.   
     
     
         102 . The method of  claim 101 , wherein determining that the antibodies can specifically bind to cells presenting the antigen peptide of interest is determined by flow cytometry. 
     
     
         103 . A method of screening an antibody that specifically binds to an antigen peptide-MHC complex, the method comprising
 exposing the animal of any one of  claims 1-89  to the antigen peptide-MHC complex comprising an antigen peptide of interest; and   sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in the cell that expresses an antibody that specifically binds to the antigen peptide-MHC complex.   
     
     
         104 . The method of  claim 103 , the method further comprises
 expressing an antibody comprising the encoding human heavy and light chain immunoglobulin variable regions;   incubating the antibody with cells presenting the antigen peptide of interest; and   determining that the antibody can specifically bind to cells presenting the antigen peptide of interest.   
     
     
         105 . The method of  claim 103 , the method further comprises
 incubating the antibody with cells presenting a control antigen peptide and/or cells do not present any antigen peptide; and   determining that the antibody cannot bind to the cells presenting the control antigen peptide or the cells that do not present any antigen peptide.